Asparagopsis algae named &#39;brominata&#39;

ABSTRACT

A novel and distinct variety of Asparagopsis taxiformis, provided as a source of halogenated compounds.

Latin name of genus and species of plant claimed: Asparagopsistaxiformis.

Varietal denomination: ‘Brominata’.

BACKGROUND INFORMATION

The present invention is a novel and distinct variety of Asparagopsistaxiformis, namely ‘Brominata’ provided as a source of halogenatedcompounds to inhibit methanogenesis in animals (e.g., ruminant animals).

Methanogenesis (i.e., the production of methane by ruminant animals) isa major contributor to global greenhouse gas emissions. Scientificliterature has demonstrated reductions in methane (CH₄) production ofbeef cattle and dairy cows when halogenated compounds are fed as part ofthe diet. See for example Roque et al., 2020: “Some haloalkanes arestructural analogs of CH₄ and therefore competitively inhibit the methyltransfer reactions that are necessary in CH₄ biosynthesis. The CH₄analogues include bromochloromethane (BCM), bromoform and chloroform andhave been proven to be the most effective feed additives for reducingCH₄ production”.

There are two potential sources for halogenated compounds: artificialsynthesis and natural production. Naturally synthesized bromoform,notably in Asparagopsis spp. has been found to mitigate a greaterpercentage of methane gas than synthetic halogenated CH₄ analogs atequivalent concentrations in vitro (Machado et. al. 2018), and tomaintain the reductions over a 147-day period in vivo (Roque, 2020).

Despite its efficacy, the potential of wild type Asparagopsis taxiformis(also referred to herein as AT) as a feed additive to inhibitmethanogenesis in ruminant animals is constrained by several factors.These include its unpleasant odor, high iodine content, epiphyticnature, and the lack of capacity, especially in male Asparagopsistaxiformis specimens, to synthesize material concentrations of thebioactive halogenated compounds.

This present invention overcomes at least some of those challengesobserved with the parent plant and optimizes the plant to synthesizebromoform. In particular, the parent plants (Asparagopsis taxiformisgametophytes) come in male and female varieties. It is only the femalevarieties that synthesize more than nominal amounts of bromoform,meaning that fifty (50%) percent of biomass is not meaninglycontributing to the overall production efficiency. With the presentinvention, not only is one hundred (100%) percent of the biomasssynthesizing bromoform, thereby almost doubling production efficiency,but the biomass of the present invention itself accumulates higherlevels of bromoform than previously attainable with female gametophytes.Further benefits of the present invention include reducing odor,reducing iodine content and rejecting epiphytes, which is beneficial tocost-effective mass production of a high-quality additive.

The novel ‘Brominata’ of the present invention is anatomicallydistinguished from the parent plant in a number of ways and wasaccomplished by a vegetative breeding program to increase bromoformconcentration.

BRIEF SUMMARY OF THE INVENTION

According to one aspect, in the present invention, we have discovered,isolated and grown a novel and distinct Asparagopsis taxiformis named‘Brominata’ with a higher bromoform concentration, lower odor, loweriodine content and higher purity than the parent plants. The features of‘Brominata’ are suitable for culture in large-scale algaculture and foruse as a cattle feed additive. This ‘Brominata’ was developed in 3phases in Kailua-Kona, Hi., USA:

-   -   i) Collection of parent plant;    -   ii) Manipulation, dissection and growth in a “seed bank” room;        and    -   iii) Selection of appropriate material from seed stock.

For the step of collecting the parent plant, wild type Asparagopsistaxiformis is collected from algal turfs or as free-floating algae inthe wild. For the step of manipulation, dissection and growth in a “seedbank” room, samples are observed and manipulated under a dissectingmicroscope to isolate, to the extent possible, clean filaments ofAsparagopsis taxiformis and separate out contaminants (e.g., epiphytes,other algae, marine animals, contaminated or unhealthy Asparagopsistaxiformis). Tiny branches are cut from the mother plant and placed insterile well plates with seawater, each well containing 360 μLof water.These samples are maintained in a “seed bank” (a room with controlledtemperature conditions, contamination protection andcarefully-calibrated light with 12-hour photoperiod daily). Cultures areregularly examined. When more than doubled in size, they are stepped-upto larger sterile well plates and then again to sterilized test tubeswith 30 ml seawater.

For the step of selecting appropriate material from seed stock, afterseven days in test tubes, material that is growing rapidly and, undermagnification, appears completely free of epiphytes and foulingorganisms is promoted to 250 ml flasks and moved to the nursery. Anadditional selection step may include selecting for promotion organismsexhibiting larger than usual gland cells. From material that has notachieved those standards, tips representing new growth are cut from thematerial, which is returned to the smallest sterile well plates withseawater, beginning the process again. The light levels and temperaturewithin the seed bank are controlled to 10-100 μE and 65-85° F. to ensuresustained growth. The growing medium is supplemented with micronutrientsin the form of F/2 medium.

As in the seedbank, material in the nursery is grown in an environmentthat is carefully maintained, including control of light (intensity,spectrum, photoperiod), temperature, micronutrients and aeration.Furthermore, flasks are aerated to ensure algae have sufficient supplyof CO₂ for photosynthesis and O₂ for respiration. Aeration also promotesmovement of biomass (beneficial to ensure access to light and preventformation of biofilm).

Throughout the process, either deep sea water or artificial seawater isused depending on the needs of the plant. Artificial seawater istypically used early in the process when plants are most vulnerable topests and diseases, and deep seawater later in the process because itcontains a rich mix of nutrients that speed plant growth.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the growth of ‘Brominata’ tetrasporophyte material in thenursery as the “filamentous” form (uniform red material in theErlenmeyer flasks, and the “puffball” form as the darker red floatingspheres;

FIG. 2 shows a closeup of the non-filamentous ‘Brominata’tetrasporophyte form;

FIG. 3 shows another closeup of the non-filamentous ‘Brominata’tetrasporophyte form;

FIG. 4 shows naturally occurring gametophytes (the parent plant). Notethe anatomical differences between ‘Brominata’ tetrasporophytes andparent gametophytes, in terms of the size and shape of the plants;

FIG. 5 shows a photomicrograph of ‘Brominata’ tetrasporophyte materialgrowing in the laboratory. Note the unusually large gland cells (theorange/black dots). The gland cells are what contains the activeingredient (bromoform). Large gland cells indicate an unusually highconcentration of bromoform;

FIG. 6 is a photomicrograph of ‘Brominata’ tetrasporophytes creatingundesired spores. Spores detract energy from growth and bromoformsynthesis.

FIG. 7 is a photo of wild ‘Brominata’ tetrasporophytes used as startingmaterial for the breeding and cultivation program.

FIG. 8 is a graph showing cell length difference between ‘Brominata’ andwild type Asparagopsis taxiformis.

FIG. 9 is a graph showing cell width difference between ‘Brominata’ andwild type Asparagopsis taxiformis.

FIG. 10 is a graph showing a volume comparison of gland cells from‘Brominata’ and wild type Asparagopsis taxiformis.

FIG. 11 is a graph showing a volume comparison of gland cells from‘Brominata’ and wild type Asparagopsis taxiformis.

FIG. 12 are pictures showing a comparison of branching in wild typeAsparagopsis taxiformis Panel A (left) and ‘Brominata’ Panel B (right).The wild type Asparagopsis taxiformis reference color patch is from theYellow-Red group of The Royal Horticultural Society Colour ChartsEdition V, while the ‘Brominata’ reference color patch is from thePurple-Blue group of the of The Royal Horticultural Society ColourCharts Edition V. The sample patches to the left of the referencepatches were derived by sampling an average color of a 5×5 pixel regionas shown by the black circle on the respective images.

FIG. 13 is a graph showing quantification of branching morphologybetween ‘Brominata’ and wild type Asparagopsis taxiformis.

DETAILED BOTANICAL DESCRIPTION

The present invention comprises the novel and distinct ‘Brominata’ thatis created through the aforementioned collection, manipulation,dissection and selection process.

The resulting ‘Brominata’ plant is a small red alga comprisingmicroscopic branched chains of cells. Unlike the gametophyte form, wherecells have differentiated functions (holdfast, stem, blades etc.), thecells in the tetrasporophyte are not highly differentiated. Instead,each cluster of four cells is roughly equivalent and these clustersstring together into long chains. The color ranges from pale pink to redto dark cherry.

Each branch contains gland cells where the bromoform is stored. Thesegland cells are a dark red to brown in color, with deeper colorindicating higher bromoform concentration.

‘Brominata’ is not rooted, but rather free-floating in water.‘Brominata’ obtains all its organic and inorganic nutrients from thewater and can live in this state indefinitely, unlike the parent plant.

‘Brominata’ is anatomically distinguished from others by stasis in thethird phase, the ‘Brominata’ tetrasporophyte phase. Wild Asparagopsistaxiformis typically follows a progression through three life stages(gametophyte, carposporophyte and tetrasporophyte). ‘Brominata’ isstatic in the tetrasporophyte phase. This is particularly beneficialbecause one hundred (100%) percent of tetrasporophytes create highlevels of bromoform, in contrast to just fifty (50%) percent ofgametophytes synthesize meaningful amounts of bromoform. In addition,since the present tetrasporophytes are static in phase they are notproducing spores. This means they can devote all of their energy togrowth, which is correlated with even higher bromoform concentrations.

Furthermore, ‘Brominata’ is special even within the tetrasporophyteclass. While tetrasporophytes, left to their own devices, devolve from“puffballs” into a filamentous form, the present ‘Brominata’tetrasporophytes can be maintained in the “puffball” phase. This isadvantageous because the ‘Brominata’ “puffball” form grows faster thanthe filamentous form. Again, this may be correlated with higherbromoform concentrations.

‘Brominata’ is not limited to the “puffballs” form but also encompassesthe larger, “cotton ball” form and the longer “filamentous” chains.

Given these anatomical differences, the composition of ‘Brominata’ ismaterially different to the parent Asparagopsis taxiformis. Inparticular, ‘Brominata’ has a much higher bromoform to iodine ratio thanthe parent Asparagopsis taxiformis. Without wishing to be bound to aparticular theory, it is believed that the lower iodine levels may, inpart, be due to high rates of bromoform synthesis and storage displacingiodine in gland cells (where bromoform is stored), as outlined in Table1 below:

TABLE 1 Comparison of Bromoform and Iodine content in Asparagopsistaxiformis Gametophyte and ‘Brominata’ Tetrasporophyte Average BromoformType of Iodine bromoform to iodine ratio Sample material (ppm) (μg/g)(μg/g: ppm) AT Beef Bag 1  Gametophyte 2265 7961 3.5 AT Beef Bag 15Gametophyte 2336 7371 3.2 AT Beef Bag 21 Gametophyte 2201 8192 3.7 WildTetrasporophyte * >67.4 **500-1500 * <<22.3 Tetrasporophyte ‘Brominata’Tetrasporophyte 67.4 9600 142.4  * Because we established that there isa strong inverse correlation between bromoform and iodine content in thetypes of algal biomass studied here, we assert that the iodine contentof the Asparagopsis tax iformis tetrasporophyte assayed here forbromoform is significantly higher than in ‘Brominata’. **This is thetypical range of bromoform that we expect would be found in wildharvested analogous Asparagopsis spp.

In addition, it is suitable for culture in vitro under illuminated andnatural light environments typical of mass production, as describedbelow.

Other distinguishing features include the taste and odor of the plant.While naturally occurring, gametophytes tend to be malodorous,‘Brominata’ tetrasporophytes have low odor. This is beneficial, sincelow-odor food tends to be more palatable.

Asparagopsis taxiformis tends to grow as epiphytes. ‘Brominata’ isdistinct because it grows as an isolated algae species. This has anumber of advantages for algal culture, including the fact that allnutrients go towards the growth of Asparagopsis taxiformis rather thancompetitive species and increased product purity.

Nevertheless, ‘Brominata’ is a fragile species, highly vulnerable topests, diseases and competitive algae. The introduction of pests orcontaminants may be prevented through a variety of mechanisms such as,but not limited to: purification cycles, maintaining positive airpressure in the flasks, using stoppers on flasks to prevent ingress ofmaterials, wearing lab coats and using shoe dips to prevent pests orcontaminants entering the lab. Furthermore, ‘Brominata’ has lowresistance to shipping or environmental changes. It can be killed orbleached by changes in temperature or light intensity. Given thissensitivity, the plant is grown under controlled environmentalconditions. Light is provided by incandescent, halogen, LED,fluorescent, high intensity discharge, metal halide, high pressuresodium or other suitable lights and maintained at 10-100 μE in the seedbank and nursery using 60-80% Blue Pearl shade cloth. Suitably filteredand controlled natural light, if available, properly filtered, and ofsufficient duration may also be used as the main, light source, or as asupplement or complement to the artificial light sources named herein,but tightly controlled artificially supplied light is preferable. Thephotoperiod is maintained at 12 hours per day to prevent sporeformation. Within the nursery, flasks are aerated to ensure algae havesufficient supply of CO₂ for photosynthesis and O₂ for respiration.Aeration also serves to promote movement of biomass. This ensures allalgae have access to light, reduces the formation of biofilm, andprevents clumping of algae, which can create an anoxic environment wherebacteria or contaminants grow. Nutrients are provided through F/2 mediumin approximately the concentrations depicted in Table 2. Temperature ismaintained at 65-85° F. (between about 18 and 30° Celsius) throughoutthe day.

TABLE 2 Concentrations of Nutrients in the F/2 Medium NutrientConcentration (ml/L) Nitrogen 6.998 Phosphate 1.500 Vitamin B1  0.053Vitamin B12 Trace Biotin Trace Iron* 0.735 Manganese* 0.026 Cobalt*0.002 Zinc* 0.003 Copper* 0.001 Molybdate* 0.001 *Only added forartificial seawater, not deep seawater

Grown under these conditions, ‘Brominata’ is a stable and uniformculture that is distinct from the parent plant. Wild type Asparagopsistaxiformis has unpleasant odor, high iodine content, epiphytic nature,and lack of capacity, especially in male specimens, to synthesizematerial concentrations of the halogenated compounds. The presentvariety has higher bromoform content, lower odor, lower iodine, anabsence of epiphytes and is static in the tetrasporophyte phase. Theseanatomically distinguishing features beneficial to cost-effective massproduction of a high-quality additive.

Photomicrographs were taken of wild type Asparagopsis taxiformis samplesand ‘Brominata’ samples. The samples were prepared by carefullyspreading a small pinch of alga on a microscope slide with tweezers,adding 2 drops of seawater, and carefully adding a cover slide. Theslide was immediately placed on the compound microscope or dissectingscope. On the compound scope, the slide was viewed through a 20× or 40×objective with a 10× eyepiece lens for a final magnification of ×200 or×400 with an illumination setting of 4. On the dissecting microscope,the slide was viewed at a magnification of 2.5× with a 10× eyepiece lensfor a final magnification of ×25. The microscope used was an OlympusCX43 with the UPlanFLN objectives. The microscope has six filtersincluding a BF (bright field), 2×, DF, Ph3, Ph2, Ph1. The dissectingscope was an Olympus SZX16 with an SDF PLAPO 1×PF objective. The filterson the microscope included BF, PO, Oblique, DF. The camera attached tothe microscope is an Olympus SC 180 camera with U-TV 0.5× cameraadapter. The computer software used to take microscope photos was acellSens Entry. Photos were saved as JPEG.

Under this brightfield photomicroscopy, ‘Brominata’ exhibits a muchdarker green signal. In a typical experiment, the following color valueswere observed: The photomicrographs were imported into Photoshop as JPEGfiles. The scale was a 397×459 pixel image at 72 pixels/inch. 5×5 pixelaverage color samples were obtained in Adobe Photoshop from the middleof a cell as shown approximately by the black circles in the respectiveimages. A 0.5×0.5 inch patch was created from the RGB values of thesamples of the wild type Asparagopsis taxiformis and ‘Brominata’ imagesand compared with The Royal Horticultural Society Colour Charts EditionV.

The wild type Asparagopsis taxiformis ‘sample patch most closely matchedpatch 50D from the Yellow-Red set, while the ‘Brominata’ derived patchmost closely matched patch N57C from the Purple-Blue group. Referencecolor patches were created from the published RGB values of The R.H.S.color chart patches and juxtaposed against the corresponding samplepatches as seen in FIG. 12. The ‘Brominata’ reference color patch had amuch lower “G” signal as compared to the wild type Asparagopsistaxiformis reference patch and having modestly lower “R” and “B” valuesas compared to the wild type Asparagopsis taxiformis reference patch aswell.

Quantitative morphology: Wild-type Asparagopsis taxiformis and‘Brominata’ were also compared quantitatively with regard to cell lengthand width, the volume of the cell to the gland cell, the branchingpattern, and the holdfast. Using the ImageJ software, the length andwidth of the cells were measured 5, 7, and 9 cells distance from theapical cell. The results are reported in FIGS. 8 and 9. The results showthat there is a significant difference in the cell length between‘Brominata’ and the wild type Asparagopsis taxiformis. A review ofpublications on Asparagopsis taxiformis found that the data collectedfrom the wild harvest material with an average length of 45 μm at themid-branch, matches that of other wild harvested material, which rangedfrom 40-65 μm) (Chualáin et al. 2004). The length of the cells in‘Brominata’ averaged a length of 20 μm at the mid-branch was shorterthan the wild type Asparagopsis taxiformis. Throughout the body of‘Brominata’, the cell and gland cell size were significantly smaller asshown in FIGS. 10-11. Branching analyses are shown in FIGS. 12-13. Theanalysis shows that ‘Brominata’ exhibits an average of around 462 μmdistance from apical cell to the first branch point versus about 248 μmfor the wild type Asparagopsis taxiformis.

1. A new and distinct variety of Asparagopsis taxiformis algae named‘Brominata’, substantially as herein illustrated and described.